Karikari, TK;
Keeling, S;
Hill, E;
Lantero Rodriguez, J;
Nagel, DA;
Becker, B;
Höglund, K;
... Moffat, KG; + view all
(2020)
An extensive plasmid library to prepare tau protein variants and study their functional biochemistry.
ACS Chemical Neuroscience
, 11
(19)
pp. 3117-3129.
10.1021/acschemneuro.0c00469.
Preview |
Text
Zetterberg_acschemneuro.0c00469.pdf - Published Version Download (4MB) | Preview |
Abstract
Tau neurofibrillary tangles are key pathological features of Alzheimer’s disease and other tauopathies. Recombinant protein technology is vital for studying the structure and function of tau in physiology and aggregation in pathophysiology. However, open-source and well-characterized plasmids for efficiently expressing and purifying different tau variants are lacking. We generated 44 sequence-verified plasmids including those encoding full length (FL) tau-441, its four-repeat microtubule-binding (K18) fragment, and their respective selected familial pathological variants (N279K, V337M, P301L, C291R, and S356T). Moreover, plasmids for expressing single (C291A), double (C291A/C322A), and triple (C291A/C322A/I260C) cysteine-modified variants were generated to study alterations in cysteine content and locations. Furthermore, protocols for producing representative tau forms were developed. We produced and characterized the aggregation behavior of the triple cysteine-modified tau-K18, often used in real-time cell internalization and aggregation studies because it can be fluorescently labeled on a cysteine outside the microtubule-binding core. Similar to the wild type (WT), triple cysteine-modified tau-K18 aggregated by progressive β-sheet enrichment, albeit at a slower rate. On prolonged incubation, cysteine-modified K18 formed paired helical filaments similar to those in Alzheimer’s disease, sharing morphological phenotypes with WT tau-K18 filaments. Nonetheless, cysteine-modified tau-K18 filaments were significantly shorter (p = 0.002) and mostly wider than WT filaments, explainable by their different principal filament elongation pathways: vertical (end-to-end) and lateral growth for WT and cysteine-modified, respectively. Cysteine rearrangement may therefore induce filament polymorphism. Together, the plasmid library, the protein production methods, and the new insights into cysteine-dependent aggregation should facilitate further studies and the design of antiaggregation agents.
| Type: | Article |
|---|---|
| Title: | An extensive plasmid library to prepare tau protein variants and study their functional biochemistry |
| Location: | United States |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1021/acschemneuro.0c00469 |
| Publisher version: | https://doi.org/10.1021/acschemneuro.0c00469 |
| Language: | English |
| Additional information: | This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. |
| Keywords: | Tau, plasmid, MAPT mutations, Alzheimer’s disease, frontotemporal dementia, expression and purification |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology > Neurodegenerative Diseases |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/10109613 |
Archive Staff Only
 |
View Item |
